Multi-scale Structural Characterization of Cellulose Microfibrils in Plant Cell Walls Using Vibrational Sum Frequency Generation (SFG) Spectroscopy

Restricted (Penn State Only)
- Author:
- Lee, Jongcheol
- Graduate Program:
- Chemical Engineering
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 04, 2024
- Committee Members:
- Andrew Zydney, Major Field Member
Enrique Gomez, Major Field Member
Seong Kim, Chair & Dissertation Advisor
Daniel Cosgrove, Outside Unit & Field Member
Robert Rioux, Professor in Charge/Director of Graduate Studies
Hojae Yi, Outside Field Member - Keywords:
- sum frequency generation
cellulose
plant cell wall
wall mechanic
X-ray diffraction
Rietveld analysis - Abstract:
- Vibrational sum frequency generation (SFG) spectroscopy is a nonlinear spectroscopy that utilizes a nonlinear optical process sensitive to vibrational dipoles in noncentrosymmetric environments. It was formerly primarily known as a ‘surface sensitive’ technique that selectively detects molecular species and their orientation at interfaces. Recently, this technique has also proven effective in selectively detecting crystalline biopolymers in natural materials without interference from other amorphous components, such as cellulose, chitin, starch, silk, and collagen. These natural materials have crystallites dispersed in amorphous phases in 3D space, and only a few vibrational dipoles from coupled vibrational modes in the crystalline phase satisfy the noncentrosymmetric selection rule, producing SFG signals. This thesis discusses the fundamentals of vibrational SFG spectroscopy that enable the analysis of polarity and structure of biopolymer crystallites in 3D space at multiple length scales, as well as its applications, focusing on the structural characterization of cellulose in natural plant cell walls. A recent development of a numerical algorithm to predict SFG responses of cellulose as a function of its orientation and structure enables proper and quantitative prediction of microfibril orientations. Plants are among the largest living organisms that can grow up to hundreds of feet tall. Plant cell walls, which surround plant cells, support their heavy weight and give plants their shape. In the walls, cellulose is known to be the major load-bearing component and is highly associated with the mechanical properties of cell walls, modulating cell expansion and growth. Thus, studying cellulose synthesis and its structure is important to understand the biological insights into plant growth, as well as the efficient utilization of cellulose as a major source of products that humans have utilized for thousands of years, such as pulp, textiles, building materials, food, and renewable biomass. A challenge in studying cellulose in cell walls is the presence of other wall components, such as pectin, hemicellulose, and lignin. These components produce interference signals in linear spectroscopies due to similar chemical moieties in their molecular structures, hinder the access of probes in scanning probe microscopies, and produce broad backgrounds in X-ray scattering, making it difficult to obtain cellulose-specific information. Another challenge in studying cellulose synthesis is the necessity of cell- or tissue-specific analysis. Cellulose synthesis is often studied through genetic mutation, and phenotypes of mutants are usually expressed in a specific tissue or a part of organs rather than in the entire plant body. Thus, to properly analyze the impact of genetic mutations, both sub-cellular scale and cellulose-specific analyses are imperative. These challenges can be overcome by vibrational SFG spectroscopy. The selective detection of crystalline cellulose provides semi-quantitative cellulose crystallinity as well as meso-scale structure of cellulose microfibrils (CMFs) in cell walls. Additionally, a femtosecond broadband laser system equipped with microscopy enables subcellular level analysis with a spatial resolution of a few micrometers, all achievable in a university laboratory setting. In this thesis, improvements in the SFG microscopy system and data processing are also presented, including the use of a tunable halfwave plate to control the infrared beam intensity, an extensometer, and fine data processing within scanned areas. Analysis of Physcomitrium patens (P. patens) leaves using SFG microscopy demonstrates a semi-quantitative analysis of crystalline cellulose and cellulose microfibril (CMF) structure in different regions in 𝜇m-scale leaves. The moss P. patens is a promising model organism for studying the cellulose synthase (CesA) protein structure-function relationship to understand cellulose synthesis. However, its small organ size has made cellulose-specific phenotype analysis challenging. SFG microscopy successfully analyzed the phenotype of a genetic mutation expressed in the leaf laminar region and the orientation of CMFs in the midrib. Plant epidermis is a widely used model system for studying cell wall mechanics to understand cell growth and cell wall enlargement. In these studies, the epidermis is often assumed to have a homogeneous cellulose microfibril structure over the entire excised cell wall fragment. However, microscopic SFG analysis has revealed preferentially aligned cellulose microfibrils (CMFs) in the cell-cell junction regions, which are distinct from the known crossed-polylamellate structure in the face region. This new discovery raises several questions: What is the possible cause of the aligned CMFs in the junction regions, and what is their contribution, as well as that of the elongated hexagonal shape of epidermal cells, to the tensile mechanical properties of tissue-scale samples? To address the complicated geometry and nonuniform material properties, finite element simulation was employed, along with microscopic SFG and cross-polarization microscopy techniques, to study the impact of the aligned CMFs in the junction regions. While SFG spectroscopy provides unique insights into cellulose structure and orientation, complementary techniques are also valuable for a comprehensive understanding of cellulose in plant cell walls. This thesis incorporates X-ray diffraction (XRD) and cross-polarization (CP) microscopy to provide a multi-faceted approach to cellulose analysis. CP microscopy, widely used for enhanced contrast in optical images and sometimes for quantification of cellulose in plant cell walls, relies on the birefringence of cellulose, which is orientation dependent. To better understand and interpret CP microscopy results, this thesis includes theoretical calculations of intensity from multiple cellulose crystals in various organizations and orientations. XRD is widely used for studying cellulose crystallinity in cell walls and cellulose-based materials. However, broad peak widths due to low ordering and background from amorphous components often complicate peak fitting, leading to inconsistent crystallinity measurements. To address this, Rietveld refinement was employed for more reliable and consistent results. This thesis also explores the crystallinity analysis of cellulose nanomaterials (CNM) using Powder X-ray Diffraction (PXRD) and the Rietveld method, as part of the VAMAS TWA 34, Project 14 initiative. It details the application of the Rietveld method for analyzing PXRD data using MAUD (Material Analysis Using Diffraction) software, with specific considerations for CNM.